Ccr1 antagonists and methods of use therefor

ABSTRACT

The invention provides compounds having the formula: 
     
       
         
         
             
             
         
       
     
     wherein R 1  is halogen. The invention also provides compositions comprising the compounds, and methods of treating diseases or disorders that comprise administering one or more of the compounds to a subject in need thereof. The disclosed compounds have CCR1 antagonist activity.

RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.11/900,744, filed on Sep. 13, 2007, which claims the benefit of U.S.patent application Ser. No. 11/204,930, filed Aug. 16, 2005, which is acontinuation of U.S. patent application Ser. No. 10/706,835, filed Nov.12, 2003, which claims the benefit of U.S. Provisional Application No.60/425,947, filed Nov. 13, 2002. The entire teachings of the aboveapplications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Chemoattractant cytokines or chemokines are a family of proinflammatorymediators that promote recruitment and activation of multiple lineagesof leukocytes, such as T lymphocytes. Chemokines can be released by manykinds of tissue cells after activation. Release of chemokines at sitesof inflammation mediates the ongoing migration of effector cells duringchronic inflammation. The chemokines are related in primary structureand contain four conserved cysteines, which form disulfide bonds. Thechemokine family includes the C—X—C chemokines (α-chemokines), and theC—C chemokines (β-chemokines), in which the first two conservedcysteines are separated by an intervening residue, or are adjacent,respectively (Baggiolini, M. and Dahinden, C. A., Immunology Today,15:127-133 (1994)).

The chemokine receptors are members of a superfamily of Gprotein-coupled receptors (GPCR) which share structural features thatreflect a common mechanism of action of signal transduction (Gerard, C.and Gerard, N. P., Annu Rev. Immunol., 12:775-808 (1994); Gerard, C. andGerard, N. P., Curr. Opin. Immunol., 6:140-145 (1994)). Conservedfeatures include seven hydrophobic domains spanning the plasma membrane,which are connected by hydrophilic extracellular and intracellularloops. The majority of the primary sequence homology occurs in thehydrophobic transmembrane regions with the hydrophilic regions beingmore diverse. The first receptor for the C—C chemokines that was clonedand expressed binds the chemokines MIP-1α and RANTES. Accordingly, thisMIP-1α/RANTES receptor was designated C—C chemokine receptor 1 (alsoreferred to as CCR-1 or CKR-1; Neote, K., et al., Cell, 72:415-425(1993); Horuk, R et al., WO 94/11504, May 26, 1994; Gao, J.-I. et al.,J. Exp. Med., 177:1421-1427 (1993)). CCR1 also binds the chemokines CCL2(MCP-1) CCL4 (MIP-1β), CCL7 (MCP-3), CCL8 (MCP-2), CCL13 (MCP-4), CCL14(HCC-1), CCL15 (Lkn-1), CCL23 (MPIF-1). (Murphy P. M. et al.,International Union of Pharmacology. XXII. Nomenclature for ChemokineReceptors, Pharmacol. Reviews, 52:145-176 (2000).) The ability ofchemokines, such as RANTES and MIP-1α, to induce the directed migrationof monocytes and a memory population of circulating T-cells indicatethat chemokines and chemokine receptors may play a critical role inchronic inflammatory diseases, since these diseases are characterized bydestructive infiltrates of T cells and monocytes. (See, e.g., Schall, T.et al., Nature, 347:669-71 (1990).)

Small molecule antagonists of the interaction between C—C chemokinereceptors (e.g., CCR1) and their ligands, including RANTES and MIP-1α,would provide compounds useful for inhibiting pathogenic processes“triggered” by receptor ligand interaction.

SUMMARY OF THE INVENTION

The invention relates to compounds having the formula:

or a physiologically acceptable salt thereof, wherein R¹ is halogen.

The invention further relates to a method for treating a diseasecharacterized by pathogenic leukocyte recruitment, pathogenic leukocyteactivation or pathogenic leukocyte recruitment and activation. Themethod comprises administering to a subject in need thereof an effectiveamount of a compound described herein.

The invention further relates to compositions comprising a compound asdescribed herein and a pharmaceutically or physiologically acceptablecarrier or excipient.

The invention further relates to the use of the compounds describedherein in therapy (including palliative, curative and prophylactictherapy) or diagnosis, and to the use of such compounds for themanufacture of a medicament for the treatment of a particular disease orcondition as described herein (e.g., inflammatory arthritis (e.g.,rheumatoid arthritis), inflammatory demyelinating disease (e.g.,multiple sclerosis)).

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to compounds that are antagonists of C—C ChemokineReceptor 1 (CCR1), compositions comprising the compounds and methods oftreating diseases or disorders that comprise administering one or moreof the compounds. The antagonist compounds can inhibit binding of aligand (e.g., a chemokine ligand such as CCL2 (MCP-1) CCL3 (MIP-1α),CCL4 (MIP-1β), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (MCP-2), CCL13 (MCP-4),CCL14 (HCC-1), CCL15 (Lkn-1), CCL23 (MPIF-1)) to CCR1. Accordingly,processes or cellular responses mediated by the binding of a chemokineto CCR1 can be inhibited (reduced or prevented, in whole or in part),including leukocyte migration, integrin activation, transient increasesin the concentration of intracellular free calcium [Ca⁺⁺]_(i), and/orgranule release of proinflammatory mediators.

The compounds have the formula:

or a physiologically acceptable salt thereof, wherein R¹ is a halogen.Preferably, the halogen is selected from the group consisting of chloro,bromo and fluoro. More preferably, the halogen is chloro.

As described herein, compounds of Formula (I) and Formula (Ia) can beprepared as racemates or as substantially pure enantiomers (>99%enantiomeric excess). The optical configuration of the compounds ofFormula (I) and Formula (Ia) are assigned using the (R),(S) method ofCahn-Ingold-Prelog. (See, J. March, “Advanced Organic Chemistry,” 4^(th)Edition, Wiley Interscience, New York, pp. 109-111 (1992).)

In preferred embodiments, the compound of Formula (I) is the(S)-enantiomer, and has the structure:

or a physiologically acceptable salt thereof, wherein R¹ is a halogen.

In a particularly preferred embodiment, the compound is of Formula IIwherein R¹ is chloro.

In other preferred embodiments, the compound of Formula (Ia) is the(S)-enantiomer, and has the structure:

or a physiologically acceptable salt thereof, wherein R¹ is a halogen.

In a particularly preferred embodiment, the compound is of Formula IIawherein R¹ is chloro.

The (S)- and (R)-enantiomers of the invention can be prepared using anysuitable method. For example, the enantiomers can be resolved from theracemate using chiral chromatography or recrystallization. Preferably,the (S)- and/or (R)-enantiomers are prepared by stereospecific synthesisas described herein.

In accordance with conventional methods for showing structural formulasof compounds, a terminal methyl group in a compound described herein canbe shown as a straight line with or without “CH₃” on its terminus:

The compounds disclosed herein can be obtained as E- andZ-configurational isomers. It is expressly pointed out that theinvention includes compounds of the E-configuration and theZ-configuration around the double bond connecting the tricyclic moietyto the remainder of the molecule, and a method of treating a subjectwith compounds of the E-configuration, the Z-configuration, and mixturesthereof. Accordingly, in the structural formulas presented herein, thesymbol:

represents both the E-configuration and the Z-configuration. Oneconfiguration can have greater activity than another. Preferably, thepyridyl ring and the piperidinyl ring are in the cis configuration asshown in Formula (II) and Formula (IIa).

The invention includes all isomeric forms and racemic mixtures of thedisclosed compounds, and a method of treating a subject with both pureisomers and mixtures thereof, including racemic mixtures.

The compounds described herein can be prepared and administered asneutral compounds, salts, esters, amides and/or prodrugs. As usedherein, “pharmaceutically or physiologically acceptable salts, esters,amides, and prodrugs” are those salts (e.g., carboxylate salts, aminoacid addition salts), esters, amides, and prodrugs of the compounds ofthe present invention which are suitable for use in contact with thetissues of a subject without undue toxicity, irritation, allergicresponse, and the like, commensurate with a reasonable benefit/riskratio, and effective for their intended use, as well as the zwitterionicforms, where possible, of the compounds of the invention.

Pharmaceutically or physiologically acceptable acid addition salts ofthe compounds described herein include salts derived from nontoxicinorganic acids such as hydrochloric, nitric, phosphoric, sulfuric,hydrobromic, hydroiodic, hydrofluoric, phosphorous, and the like, andsalts derived from nontoxic organic acids, such as aliphatic mono- anddicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoicacids, alkanedioic acids, aromatic acids, aliphatic and aromaticsulfonic acids, and the like. Such acid additional salts include, forexample, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate,phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide, acetate, trifluoroacetate,propionate, caprylate, isobutyrate, oxalate, malonate, succinate,suberate, sebacate, fumarate, maleate, mandelate, benzoate,chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate,benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate,maleate, tartrate and methanesulfonate salts. Also contemplated aresalts of amino acids such as arginate, gluconate, galacturonate and thelike. (See, for example, Berge S. M. et al., “Pharmaceutical Salts,” J.Pharma. Sci., 66:1 (1977).)

Acid addition salts of compounds which contain a basic group (e.g.,amine) can be prepared using suitable methods. For example, acidaddition salts can be prepared by contacting the free base form of acompound with a sufficient amount of a desired acid to produce the saltin the conventional manner. The free base form can be regenerated bycontacting the salt form with a base and isolating the free base in theconventional manner. The free base form of a compound can differ from asalt forms somewhat in certain physical properties such as solubility inpolar solvents.

Pharmaceutically or physiologically acceptable base addition salts canbe formed with suitable metals or amines, such as alkali and alkalineearth metals or organic amines. Examples of metals which are suitablefor use as cations in base addition salts include sodium, potassium,magnesium, calcium and the like. Amines suitable for use as cations inbase addition salts include N,N′-dibenzylethylenediamine,chloroprocaine, choline, diethanolamine, dicyclohexylamine,ethylenediamine, N-methylglucamine, and procaine. (See, for example,Berge S. M. et al., “Pharmaceutical Salts,” J. Pharma. Sci., 66:1(1977).)

Base addition salts of compounds which contain an acidic group (e.g.,carboxylic acid) can be prepared using suitable methods. For example,the free acid form of a compound can be contacted with a sufficientamount of the desired base to produce a salt in the conventional manner.The free acid form can be regenerated by contacting the salt form with asuitable acid and isolating the free acid in the conventional manner.The free acid form of a compound can differ from the base addition saltform somewhat in certain physical properties such as solubility in polarsolvents.

The term “prodrug” refers to compounds that can be transformed in vivo(e.g., following administration to an animal), by metabolic processes orother processes, to yield a compound of the above formulae, for example,by hydrolysis in blood. A thorough discussion is provided in T. Higuchiand V. Stella, “Pro-drugs as Novel Delivery Systems,” Vol. 14 of theA.C.S. Symposium Series; and Bioreversible Carriers in Drug Design, ed.Edward B. Roche, American Pharmaceutical Association and Pergamon Press,1987, both of which are incorporated herein by reference. Suitableprodrugs include pharmaceutically or physiologically acceptable estersand amides of the compounds described herein. Examples ofpharmaceutically or physiologically acceptable, esters of the compoundsof this invention include C₁-C₆ alkyl esters. In certain embodiments,the alkyl group of the alkyl ester is a straight or branched chain C₁-C₆alkyl group. Acceptable alkyl esters also include C₅-C₇ cycloalkylesters as well as arylalkyl esters such as, but not limited to benzyl.C₁-C₄ esters are preferred. Esters of the compounds of the presentinvention can be prepared using any suitable method.

Examples of pharmaceutically or physiologically acceptable, amides ofthe compounds of this invention include amides derived from ammonia,primary C₁-C₆ alkyl amines and secondary C₁-C₆ dialkyl amines whereinthe alkyl groups are straight or branched chain. In the case ofsecondary amines, the amine may also be in the form of a 5- or6-membered heterocycle containing one nitrogen atom. Amides derived fromammonia, C₁-C₃ alkyl primary amines, and C₁-C₂ dialkyl secondary aminesare preferred. Amides of the compounds of the invention may be preparedusing any suitable method.

Compositions

The invention also relates to pharmaceutical and/or physiologicalcompositions which contain one or more of the compounds describedherein. Such compositions can be formulated for administration by anydesired route, such as orally, topically, by inhalation (e.g.,intrabronchial, intranasal, oral inhalation or intranasal drops),rectally, transdermally, or parenterally. Generally the compositionscomprise a compound of the invention (i.e., one or more compounds) asthe active ingredient and a (one or more) suitable carrier, diluent,excipient, adjuvant and/or preservative. Formulation of a compound to beadministered will vary according to the route of administration selected(e.g., solution, emulsion, capsule). Standard pharmaceutical formulationtechniques can be employed. (See, generally, “Remington's PharmaceuticalScience,” 18^(th) Edition, Mack Publishing. (1990); Baker, et al.,“Controlled Release of Biological Active Agents,” John Wiley and Sons(1986), the entire teachings of both of the foregoing are incorporatedherein by reference.)

The presence of microorganisms in the compositions can be controlled byvarious antibacterial and/or antifungal agents, for example, parabens,chlorobutanol, alcohols (e.g., phenol, benzyl alcohol), sorbic acid, andthe like. It may also be desirable to include isotonic agents, forexample sugars, sodium chloride, and the like.

Compositions suitable for parenteral injection can comprisephysiologically acceptable sterile aqueous or nonaqueous solutions,dispersions, suspensions or emulsions, and sterile powders forreconstitution into sterile injectable solutions or dispersions.Examples of suitable aqueous and nonaqueous carriers, diluents,solvents, excipients or vehicles include physiological saline,phosphate-buffered saline, Hank's solution, Ringer's-lactate and thelike, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol,and the like), vegetable oils (such as olive oil) and injectable organicesters such as ethyl oleate, or any suitable mixture thereof. Fluiditycan be adjusted, for example, by the use of a coating such as lecithin,by the maintenance of the required particle size in the case ofdispersions and by the use of surfactants. When prolonged absorption ofan injectable pharmaceutical composition is desired, agents that delayabsorption, for example, aluminum monostearate and gelatin can beincluded.

Solid dosage forms for oral administration include, for example,capsules, tablets, pills, powders, and granules. In such solid dosageforms, the active ingredient (i.e., one or more compounds of theinvention) can be admixed with one or more carrier or excipient such assodium citrate or dicalcium phosphate; (a) fillers or extenders, forexample, starches, lactose, sucrose, glucose, mannitol, silicic acid,polyethyleneglycols, and the like; (b) binders, for example,carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone,sucrose, and acacia; (c) humectants, for example, glycerol; (d)disintegrating agents, for example, agar-agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain complex silicates, and sodiumcarbonate; (e) solution retarders, for example paraffin; (f) absorptionaccelerators, for example, quaternary ammonium compounds; (g) wettingagents, for example, cetyl alcohol, and glycerol monostearate; (h)adsorbents, for example, kaolin and bentonite; and (i) lubricants, forexample, talc, calcium stearate, magnesium stearate, solid polyethyleneglycols, sodium lauryl sulfate, or mixtures thereof. Solid compositions,such as those for oral administration, can also comprise bufferingagents. Such solid compositions or solid compositions that are similarto those described can be provided in soft- or hard-filled gelatincapsules if desired.

Solid dosage forms such as tablets, dragées, capsules, pills, andgranules can be prepared with coatings and shells, such as entericcoatings or other suitable coatings or shells. Several such coatingand/or shells are well known in the art, and can contain opacifyingagents, and can also be of such composition that they release the activecompound or compounds in a certain part of the intestinal tract in adelayed manner. Examples of embedding compositions which can be used arepolymeric substances and waxes. The active compounds can also be used inmicroencapsulated form, if appropriate, with, for example, one or moreof the above-mentioned carriers or excipients.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirs. Inaddition to the active compounds, the liquid dosage forms can contain asuitable carrier or excipient, such as water or other solvents,solubilizing agents and emulsifiers, as for example, ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propyleneglycol, 1,3-butyleneglycol, dimethylformamide,oils, in particular, cottonseed oil, groundnut oil, corn germ oil, oliveoil, castor oil and sesame oil, glycerol, tetrahydrofurfuryl alcohol,polyethyleneglycols and fatty acid esters of sorbitan or mixtures ofthese substances, and the like. If desired, the composition can alsoinclude wetting agents, emulsifying agents, suspending agents,sweetening, flavoring and/or perfuming agents. Suspensions can containsuspending agents, such as, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth, andthe like. Mixtures of suspending agents can be employed if desired.Suppositories (e.g., for rectal or vaginal administration) can beprepared by mixing one or more compounds of the invention with suitablenonirritating excipients or carriers such as cocoa butter,polyethyleneglycol, or a suppository wax which is solid at roomtemperature but liquid at body temperature and melts in the rectum orvagina, thereby releasing the active ingredient.

Dosage forms for topical administration include ointments, powders,sprays and inhalants. The active ingredient can be admixed undersuitable conditions (e.g., sterile conditions) with a physiologicallyacceptable carrier and any preservatives, buffers, or propellants as maybe required. Ophthalmic formulations, eye ointments, powders, andsolutions can also be prepared, for example, using suitable carriers orexcipients. For inhalation, the compound can be solubilized and loadedinto a suitable dispenser for administration (e.g., an atomizer,nebulizer or pressurized aerosol dispenser).

The quantity of active ingredient (one or more compounds of theinvention) in the composition can range from about 0.1% to about 99.9%by weight. Preferably the quantity of active ingredient is about 10% toabout 90%, or about 20% to about 80% by weight. A unit dose preparationcan contain from 1 mg to about 1000 mg active ingredient, preferablyabout 10 mg to about 100 mg active ingredient. The composition can, ifdesired, also contain other compatible therapeutic agents, such astheophylline, ÿ-adrenergic bronchodilators, corticosteroids,antihistamines, antiallergic agents, immunosuppressive agents (e.g.,cyclosporin A, FK-506, prednisone, methylprednisolone), hormones (e.g.,adrenocorticotropic hormone (ACTH)), cytokines (e.g., interferons (e.g.,IFNβ-1a, IFNβ-1b)) and the like.

In one embodiment, the composition comprises(S)-5-{3-[4-(4-Chloro-phenyl)-4-hydroxy-3,3-dimethyl-piperidin-1-yl]-propylidene}-5,11-dihydro-10-oxa-1-aza-dibenzo[a,d]cycloheptene-7-carboxylicacid and a physiologically acceptable carrier or excipient. In anotherembodiment, the composition is substantially free of(R)-5-{3-[4-(4-Chloro-phenyl)-4-hydroxy-3,3-dimethyl-piperidin-1-yl]-propylidene}-5,11-dihydro-10-oxa-1-aza-dibenzo[a,d]cycloheptene-7-carboxylicacid (contains at least about 98% or at least about 99% enantiomericexcess of(S)-5-{3-[4-(4-Chloro-phenyl)-4-hydroxy-3,3-dimethyl-piperidin-1-yl]-propylidene}-5,11-dihydro-10-oxa-1-aza-dibenzo[a,d]cycloheptene-7-carboxylicacid).

In another embodiment, the composition comprises(S)-5-{3-[4-(4-Chloro-phenyl)-4-hydroxy-3,3-dimethyl-piperidin-1-yl]-propylidene}-5,11-dihydro-10-oxa-1-aza-dibenzo[a,d]cycloheptene-7-carboxylic acid,(R)-5-{3-[4-(4-Chloro-phenyl)-4-hydroxy-3,3-dimethyl-piperidin-1-yl]-propylidene}-5,11-dihydro-10-oxa-1-aza-dibenzo[a,d]cycloheptene-7-carboxylicacid and a physiologically acceptable carrier or excipient. In oneembodiment, the composition comprisesracemic-5-{3-[4-(4-Chloro-phenyl)-4-hydroxy-3,3-dimethyl-piperidin-1-yl]-propylidene}-5,11-dihydro-10-oxa-1-aza-dibenzo[a,d]cycloheptene-7-carboxylicacid In other embodiments, the ratio (S)-enantiomer:(R)-enantiomer (w/w)is at least about 2:1 or about 5:1 or about 10:1 or about 20:1 or about50:1.

In one embodiment, the composition comprises(S)-5-{3-[4-(4-Chloro-phenyl)-4-hydroxy-3,3-dimethyl-piperidin-1-yl]-propylidene}-1-oxy-5,11-dihydro-10-oxa-1-aza-dibenzo[a,d]cycloheptene-7-carboxylicacid and a physiologically acceptable carrier or excipient. In anotherembodiment, the composition is substantially free of(R)-5-{3-[4-(4-Chloro-phenyl)-4-hydroxy-3,3-dimethyl-piperidin-1-yl]-propylidene}-1-oxy-5,11-dihydro-10-oxa-1-aza-dibenzo[a,d]cycloheptene-7-carboxylicacid (contains at least about 98% or at least about 99% enantiomericexcess of(S)-5-{3-[4-(4-Chloro-phenyl)-4-hydroxy-3,3-dimethyl-piperidin-1-yl]-propylidene}-1-oxy-5,11-dihydro-10-oxa-1-aza-dibenzo[a,d]cycloheptene-7-carboxylicacid).

In another embodiment, the composition comprises(S)-5-{3-[4-(4-Chloro-phenyl)-4-hydroxy-3,3-dimethyl-piperidin-1-yl]-propylidene}-1-oxy-5,11-dihydro-10-oxa-1-aza-dibenzo[a,d]cycloheptene-7-carboxylicacid,(R)-5-{3-[4-(4-Chloro-phenyl)-4-hydroxy-3,3-dimethyl-piperidin-1-yl]-propylidene}-1-oxy-5,11-dihydro-10-oxa-1-aza-dibenzo[a,d]cycloheptene-7-carboxylicacid and a physiologically acceptable carrier or excipient. In oneembodiment, the composition comprisesracemic-5-{3-[4-(4-Chloro-phenyl)-4-hydroxy-3,3-dimethyl-piperidin-1-yl]-propylidene}-1-oxy-5,11-dihydro-10-oxa-1-aza-dibenzo[a,d]cycloheptene-7-carboxylicacid In other embodiments, the ratio (S)-enantiomer:(R)-enantiomer (w/w)is at least about 2:1 or about 5:1 or about 10:1 or about 20:1 or about50:1.

Therapeutic Methods

The invention further relates to a method for treating (e.g.,palliative, curative, prophylactic) a disease or disorder associatedwith pathogenic leukocyte recruitment, activation or recruitment andactivation, mediated by chemokines or chemokine receptor functionincluding chronic and acute inflammatory disorders.

As used herein “pathogenic leukocyte recruitment, activation orrecruitment and activation” refers to leukocyte recruitment (e.g.,accumulation of leukocytes at a sight of inflammation or injury) and/oractivation (e.g., physiologic state in which leukocytes perform effectorfunctions) that contributes to the conditions, processes or results ofthe disease or disorder to be treated. For example, in a subjectafflicted with multiple sclerosis, recruitment and/or activation of Tcells in the central nervous system is considered “pathogenic leukocyterecruitment, pathogenic leukocyte activation or pathogenic leukocyterecruitment and activation,” because recruited and activated T cellscontribute to the demyelination characteristic of that disease.Similarly, in a subject afflicted with rheumatoid arthritis, recruitmentand/or activation of T cells in joints (e.g., synovial tissue or fluid)is considered “pathogenic leukocyte recruitment, pathogenic leukocyteactivation or pathogenic leukocyte recruitment and activation,” becauserecruited and activated T cells contribute to the tissue destructioncharacteristic of rheumatoid arthritis.

Diseases and disorders characterized by pathogenic leukocyterecruitment, pathogenic leukocyte activation or pathogenic leukocyterecruitment and activation that can be treated according to the methodsdescribed herein include, for example, acute and chronic inflammatorydisorders characterized by the presence of CCL2 (MCP-1) CCL3 (MIP-1α),CCL4 (MIP-1β), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (MCP-2), CCL13 (MCP-4),CCL14 (HCC-1), CCL15 (Lkn-1) and/or CCL23 (MPIF-1) responsive cells,such as T cells, monocytes or eosinophils. Such diseases or disordersinclude, but are not limited to, inflammatory arthritis (e.g.,rheumatoid arthritis), inflammatory demyelinating disease (e.g.,multiple sclerosis), atherosclerosis, arteriosclerosis, restenosis,ischemia/reperfusion injury, diabetes mellitus (e.g., type 1 diabetesmellitus), psoriasis, inflammatory bowel diseases such as ulcerativecolitis and Crohn's disease, rejection (acute or chronic) oftransplanted organs and tissues (e.g., acute allograft rejection,chronic allograft rejection), graft versus host disease, as well asallergies and asthma. Other diseases associated with aberrant leukocyterecruitment and/or activation which can be treated (includingprophylactic treatments) with the methods disclosed herein areinflammatory diseases associated with viral (e.g., HumanImmunodeficiency Virus (HIV)), bacterial or fungal infection, such as,AIDS associated encephalitis, AIDS related maculopapular skin eruption,AIDS related interstitial pneumonia, AIDS related enteropathy, AIDSrelated periportal hepatic inflammation and AIDS related glomerulonephritis. The method comprises administering to the subject in need oftreatment an effective amount of a compound (i.e., one or morecompounds) described herein.

As used herein “inflammatory demyelinating disease” refers to acute andchronic inflammatory diseases characterized by demyelination of centralnervous system tissue. The inflammatory demyelinating disease can be anacute inflammatory demyelinating disease, for example, acutedisseminated encephalomyelitis, Guillain-Barre syndrome or acutehemorrhagic leukoencephalitis. In other embodiments, the inflammatorydemyelinating disease can be a chronic inflammatory demyelinatingdisease, for example, multiple sclerosis, chronic inflammatorydemyelinating polyradiculoneuropathy.

In a preferred embodiment, the invention provides a method of treatingmultiple sclerosis, comprising administering an effective amount of acompound of Formula (I), (Ia), (II) or (IIa) to a subject in needthereof. The manifestation of MS is variable and the clinical course ofMS can be grouped into four categories: relapsing-remitting, primaryprogressive, secondary progressive and progressive-relapsing. The methodof the invention can be used to treat MS which presents with each of therecognized clinical courses. Accordingly, a compound of the inventioncan be administered to a patient with a progressive course of MS toretard or prevent the progression of neurological impairment. A compoundof the invention can also be administered to a subject withrelapsing-remitting, secondary progressive or progressive-relapsing MSto inhibit relapse (e.g., an acute attack). For example, a compound ofthe invention can be administered to a subject with relapsing-remittingMS during the remitting phase of the disease to prevent or delayrelapse.

As used herein, “inflammatory arthritis” refers to those diseases ofjoints where the immune system is causing or exacerbating inflammationin the joint, and includes rheumatoid arthritis, juvenile rheumatoidarthritis and spondyloarthropathies, such as ankylosing spondylitis,reactive arthritis, Reiter's syndrome, psoriatic arthritis, psoriaticspondylitis, enteropathic arthritis, enteropathic spondylitis,juvenile-onset spondyloarthropathy and undifferentiatedspondyloarthropathy. Inflammatory arthritis is generally characterizedby infiltration of the synovial tissue and/or synovial fluid byleukocytes.

In another preferred embodiment, the invention provides a method oftreating rheumatoid arthritis, comprising administering an effectiveamount of a compound of Formula (I), (Ia), (II) or (IIa) to a subject inneed thereof.

A “subject” is preferably a bird or mammal, such as a human (Homosapiens), but can also be an animal in need of veterinary treatment,e.g., domestic animals (e.g., dogs, cats, and the like), farm animals(e.g., cows, sheep, fowl, pigs, horses, and the like) and laboratoryanimals (e.g., rats, mice, guinea pigs, and the like).

An “effective amount” of a compound is an amount which inhibits bindingof chemokine to receptor (e.g., CCR1) and thereby inhibits one or moreprocesses mediated by the binding in a subject with a disease associatedwith pathogenic leukocyte recruitment, pathogenic leukocyte activationor pathogenic leukocyte recruitment and activation. Examples of suchprocesses include leukocyte migration, integrin activation, transientincreases in the concentration of intracellular free calcium [Ca²⁺]_(i)and granule release of proinflammatory mediators. An “effective amount”of a compound can achieve a desired therapeutic and/or prophylacticeffect, such as an amount which results in the prevention of or adecrease in the symptoms associated with a disease associated withpathogenic leukocyte recruitment, pathogenic leukocyte activation orpathogenic leukocyte recruitment and activation.

The amount of compound administered to the individual will depend on thetype and severity of the disease and on the characteristics of theindividual, such as general health, age, sex, body weight and toleranceto drugs. It will also depend on the degree, severity and type ofdisease. The skilled artisan will be able to determine appropriatedosages depending on these and other factors. An antagonist of chemokinereceptor function can also be administered in combination with one ormore additional therapeutic agents, such as, theophylline, β-adrenergicbronchodilators, corticosteroids, antihistamines, antiallergic agents,immunosuppressive agents (e.g., cyclosporin A, FK-506, prednisone,methylprednisolone), hormones (e.g., adrenocorticotropic hormone(ACTH)), cytokines (e.g., interferons (e.g., IFNβ-1a, IFNβ-1b)) and thelike.

When a compound of the invention is administered in combination withanother therapeutic agent, the compound and agent can be administered ina manner that afford overlap of pharmacological activity, for example,concurrently or sequentially.

The compound can be administered by any suitable route, including, forexample, orally in capsules, suspensions or tablets or by parenteraladministration. Parenteral administration can include, for example,systemic administration, such as by intramuscular, intravenous,subcutaneous or intraperitoneal injection. The compound can also beadministered orally (e.g., dietary), transdermally, topically, byinhalation (e.g., intrabronchial, intranasal, oral inhalation orintranasal drops), or rectally, depending on the disease or condition tobe treated. Oral or parenteral administration are preferred modes ofadministration. The compound can be administered to the individual aspart of a pharmaceutical or physiological composition.

The activity of compounds of the present invention can be assessed usingsuitable assays, such as receptor binding assays or chemotaxis assays.For example, as described in the Examples, small molecule antagonists ofMIP-1ÿ binding have been identified utilizing THP-1 cells membranes.Specifically, a high through-put receptor binding assay, which monitors¹²⁵I-MIP-1α binding to THP-1 cell membranes, was used to identify smallmolecule antagonists which block binding of MIP-1α. Compounds of thepresent invention can also be identified by virtue of their ability toinhibit the activation steps triggered by binding of a chemokine (e.g.,CCL2 (MCP-1) CCL3 (MIP-1α), CCL4 (MIP-1β), CCL5 (RANTES), CCL7 (MCP-3),CCL8 (MCP-2), CCL13 (MCP-4), CCL14 (HCC-1), CCL15 (Lkn-1), CCL23(MPIF-1)) to its receptor (CCR-1), such as chemotaxis, integrinactivation and granule mediator release. They can also be identified byvirtue of their ability to block chemokine (e.g., CCL2 (MCP-1) CCL3(MIP-1α), CCL4 (MIP-1β), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (MCP-2),CCL13 (MCP-4), CCL14 (HCC-1), CCL15 (Lkn-1), CCL23 (MPIF-1)) inducedchemotaxis of, for example, HL-60 cells, T-cells, peripheral bloodmononuclear cells or eosinophils.

EXAMPLES

Example 1

(S)-5-{3-[4-(4-Chloro-phenyl)-4-hydroxy-3,3-dimethyl-piperidin-1-yl]-propylidene}-5,11-dihydro-10-oxa-1-aza-dibenzo[a,d]cycloheptene-7-carboxylicacid Step 1: 3,3-Dimethyl-4-oxo-piperidine-1-carboxylic acid tert-butylester

To a dry, 2 L 2-neck, round-bottom flask equipped with a magneticstirrer, a condenser, and a large 10° C. water bath was added4-oxo-piperidine-1-carboxylic acid tert-butyl ester (125 g, 628 mmol)and anhydrous tetrahydrofuran (1 L). To the resulting yellow solutionwas added methyl iodide (85 mL, 1365 mmol). Sodium t-butoxide (150 g,1560 mmol) was then added portionwise over 30 minutes. An exotherm wasdetected, especially at the beginning of the addition. The reactionmixture did warm to a gentle reflux, the rate was controlled by thespeed of addition of base. The mixture was stirred an additional 30minutes. The solvent was removed in vacuo. The oily residue was treatedwith NH₄Cl/water (500 mL), and extracted with ether (3×200 mL). Thecombined organics were washed with brine, dried over Na₂SO₄, andfiltered through a short plug of silica gel. The solvent was removed invacuo, and the resulting yellow oil had started to crystallize. It wasleft under high vacuum overnight. The mixture was slurried in hexane(50-100 mL) and sonicated for one minute. The yellow solid was collectedby filtration and washed with hexane (100 mL). The first crop of3,3-dimethyl-4-oxo-piperidine-1-carboxylic acid tert-butyl ester yieldeda yellow solid. (See, preparation of (37) in Vice, S. et al., J. Org.Chem., 66:2487-2492 (2001).)

¹H-NMR (CDCl₃, 300 MHz) δ: 1.13 (s, 6H), 1.49 (s, 9H), 2.49 (t, 2H),3.43 (br s, 2H), 3.73 (t, 2H).

Step 2:4-(4-Chloro-phenyl)-4-hydroxy-3,3-dimethyl-piperidine-1-carboxylic acidtert-butyl ester

A 2-neck, 2-L round bottom flask was fitted with two 125 mL droppingfunnels and a stir bar. The assembly was flame-dried under dry nitrogen.The flask was charged with THF (700 mL) and 4-bromo-chlorobenzene (33.7g, 176 mmol, 2.5 eq.). The resulting solution was cooled to −78° C. in adry ice/acetone bath. To one of the dropping funnels was addedbutyllithium (2.5 M in hexanes, 70 mL, 175 mmol, 2.5 eq) via canula. Thebutyllithium solution was slowly added to the cold THF solution over 1hour. Stirring continued for an additional 0.5 hour affording a whitesuspension. A solution of 3,3-dimethyl-4-oxo-piperidine-1-carboxylicacid tert-butyl ester (16.0 g, 70.5 mmol, 1 eq.) in THF (100 mL) wasprepared and added to the reaction mixture via the second droppingfunnel over 1.75 hours. The resulting mixture was stirred at −78° C. for2 hours, at which time the reaction appeared to be essentially completeby TLC analysis. Saturated aqueous NH₄Cl (150 mL) was added and thereaction was allowed to warm to room temperature. Water (150 mL) wasadded and the mixture was extracted with ethyl acetate (2+1 L). Thecombined extracts were washed with water and brine, dried over magnesiumsulfate, filtered and concentrated. The solid residue was trituratedwith ethyl acetate and filtered. The supernatant was concentrated andtriturated with ether. The resulting supernatant was then trituratedwith ether/petroleum ether. The resulting solids were combined to afford4-(4-Chloro-phenyl)-4-hydroxy-3,3-dimethyl-piperidine-1-carboxylic acidtert-butyl ester as an off-white solid.

¹H-NMR (CDCl₃, 300 MHz) δ: 0.82 (s, 6H), 1.34-1.44 (m, 2H), 1.49 (s,9H), 2.67 (ddd, 1H), 3.10-3.70 (m, 3H), 4.00-4.30 (m, 1H), 7.31 (d, 2H),7.39 (d, 2H).

Step 3: 4-(4-Chloro-phenyl)-3,3-dimethyl-piperidin-4-ol

To a cooled (0° C.) solution of4-(4-Chloro-phenyl)-4-hydroxy-3,3-dimethyl-piperidine-1-carboxylic acidtert-butyl ester (10.42 g, 30.7 mmol) in methylene chloride (300 mL) wasslowly added trifluoroacetic acid (60 mL) over 1.25 hours. The resultingyellow solution was stirred at 0° C. for an additional 1.5 hours. Themixture was concentrated under reduced pressure and the residuedissolved in ethyl acetate (1.2 L), and washed with aqueous sodiumhydroxide (1 N, 150 mL). The aqueous layer was extracted with additionalethyl acetate (200 mL) and the combined extracts were washed with brine,dried over sodium sulfate, filtered and concentrated. The resultingsolid residue was triturated with ether to afford4-(4-Chloro-phenyl)-3,3-dimethyl-piperidin-4-ol as an off-white solid.

¹H-NMR (CD₃OD, 300 MHz) δ: 0.73 (s, 3H), 0.85 (s, 3H), 1.42 (ddd, 1H),2.36 (d, 1H), 2.61 (ddd, 1H), 2.91 (br dd, 1H), 3.08-3.19 (m, 2H),7.26-7.32 (m, 2H), 7.44-7.50 (m, 2H).

MS m/z: 240 (M+1).

Step 4: (S)-4-(4-chloro-phenyl)-3,3-dimethyl-piperidin-4-ol

A visibly clean 5 L, 3-neck flask was fitted with an overhead stirrerand flushed with nitrogen for 20 min. Racemic4-(4-Chloro-phenyl)-3,3-dimethyl-piperidin-4-ol (202 g, 843 mmol),L-(+)-tartaric acid (114 g, 759 mmol) and 4040 mL of a 9:1butanone:water mixture were added to the flask. The mixture was heatedto reflux. Water (202 mL) was added portionwise over 45 min (ratio ofbutanone to water: 6:1) to fully dissolve the solid mixture. Reflux wascontinued an additional 45 minutes, the heat source was then turned offand the flask allowed to cool slowly to room temperature overnight.Solids were removed under suction filtration and dried for about 3 daysin vacuo to afford S-enantiomer as the L-(+) tartrate salt, which waspartitioned between 1 M NaOH and methylene chloride (brine washed andsodium sulfate-dried) to afford the free base.

¹H-NMR (CD₃OD, 300 MHz) δ: 0.73 (s, 3H), 0.85 (s, 3H), 1.42 (ddd, 1H),2.36 (d, 1H), 2.61 (ddd, 1H), 2.91 (br dd, 1H), 3.08-3.19 (m, 2H),7.26-7.32 (m, 2H), 7.44-7.50 (m, 2H).

MS m/z: 240 (M+1).

Step 5: 5-Cyclopropyl-5,11-dihydro[1]benzoxepino[2,3-b]pyridin-5-ol

A dry 2 L three-necked, round-bottomed flask was fitted with a magneticstirring bar, a glass stopper, a rubber septum, and an argon inlet.Under an argon atmosphere, 50.0 g. of5,11-dihydro[1]benzoxepino[3,4-b]pyridine-5-one (prepared by the methodof Inoue et al., Synthesis 1:113-116 (1997), (0.24 mole)) and 500 mL ofdry tetrahydrofuran were added to the flask and the flask was cooledwith an ice bath. A freshly prepared cyclopropylmagnesium bromidetetrahydrofuran solution (50.0 g. of cyclopropylmagnesium bromide wasprepared from cyclopropylbromide (0.41 mole) and 12.0 g. of magnesiumturnings (0.49 mole) in 400 mL of dry tetrahydrofuran) was introduced byneedle over a period of 5 minutes. The ice bath was removed, and themixture was stirred for 30 minutes. The reaction mixture was slowlypoured into 500 mL of saturated ammonium chloride solution, the mixturewas extracted with two 300 mL portions of ethyl acetate, and thecombined organic extracts are washed with 300 mL of saturated aqueoussodium chloride. The organic solution was dried with anhydrous magnesiumsulfate, filtered, and evaporated (aspirator vacuum, ca. 30° C.). To theresidual solid was added 150 mL of a 1:1 (v/v) hexane-ethyl acetatemixture, and the mixture was sonicated for 15 minutes, filtered andwashed with a 1:1 (v/v) hexane-ethyl acetate mixture to yield the titledcompound as a pale yellow solid.

Step 6: 5-(3-Bromopropylidene)-5,11-dihydro[1]benzoxepino[2,3-b]pyridine

To a 2 L eggplant flask with a magnetic stirring bar was added 75.0 g.of 5-Cyclopropyl-5,11-dihydro[1]benzoxepino[2,3-b]pyridin-5-ol (0.30mole) and 75 mL of acetic acid. The solution was cooled with water (ca.10° C.), 120 ml of 47% aqueous hydrobromic acid was added over a periodof 5 minutes. The reaction mixture was warmed to 60° C., stirred for anhour, and evaporated (aspirator vacuum, ca. 50° C.) to ca. 200 mL. Thereaction mixture was poured to 1500 mL of saturated aqueous sodiumbicarbonate, the mixture is extracted with two 800 mL portions of ethylacetate, and the combined organic extracts are washed with 500 mL ofsaturated aqueous sodium chloride. The organic solution was dried withanhydrous magnesium sulfate, filtered, and evaporated (aspirator vacuum,ca. 30° C.). The oily residue was chromatographed on 500 g. of Silicagel 60 by eluting with 5:1-4:1 (v/v) hexane-ethyl acetate mixture. Theelution was evaporated, giving the titled compound as a pale yellow oil.

Step 7:7-Acetyl-5-(3-bromopropylidene)-5,11-dihydro[1]benzoxepino[2,3-b]pyridine

A dry 3 L three-necked, round-bottomed flask was fitted with a magneticstirring bar, a glass stopper, a rubber septum, and an argon inlet.Under an argon atmosphere, 94.0 g. of5-(3-Bromopropylidene)-5,11-dihydro[1]benzoxepino[2,3-b]pyridine (0.30mole) and 900 mL of dry dichloromethane were added to the flask and theflask was cooled with an ice bath. To the solution was slowly added 78.5g. of aluminum chloride (0.83 mole), followed by 17.8 mL of acetylchloride (0.25 mole), and the mixture was stirred for an hour at 0° C.The reaction mixture was poured to 1500 g of ice, and the layers wereseparated. The aqueous layer was extracted with three 400 mL portions ofethyl acetate. Dichloromethane layer and the organic extracts werecombined and washed successively with 1 μL of saturated aqueous sodiumbicarbonate and 1 L of saturated aqueous sodium chloride. The organicsolution was dried with anhydrous magnesium sulfate, filtered, andevaporated (aspirator vacuum, ca. 30° C.). The oily residue waschromatographed on 800 g. of Silica gel 60 by eluting with 5:1-1:1 (v/v)hexane-ethyl acetate mixture. The elution was evaporated, giving thetitled compound as a pale yellow solid.

Step 8:(S)-1-(5-{3-[4-(4-Chloro-phenyl)-4-hydroxy-3,3-dimethyl-piperidin-1-yl]-propylidene}-5,11-dihydro-10-oxa-1-aza-dibenzo[a,d]cyclohepten-7-yl)-ethanone

To a suspension of the(S)-4-(4-chloro-phenyl)-3,3-dimethyl-piperidin-4-ol (5.50 g, 22.94 mmol)in acetonitrile (200 mL) and water (50 mL) was added potassium carbonate(7.17 g, 51.9 mmol) followed by solid1-[5-(3-bromo-propylidene)-5,11-dihydro-10-oxa-1-aza-dibenzo[a,d]cyclohepten-7-yl]-ethanone(6.30 g, 17.3 mmol). The heterogeneous mixture was stirred at roomtemperature 4 hours, warmed to 50° C. and stirred 13 hours. The mixturewas cooled to room temperature and acetonitrile was removed underreduced pressure. The aqueous layer was extracted with ethyl acetate(750 mL) and the extract was washed with brine, dried over sodiumsulfate, filtered and concentrated. The crude residue was purified bysilica gel chromatography (3:1 ethyl acetate:hexanes) to afford(S)-1-(5-{3-[4-(4-Chloro-phenyl)-4-hydroxy-3,3-dimethyl-piperidin-1-yl]-propylidene}-5,11-dihydro-10-oxa-1-aza-dibenzo[a,d]cyclohepten-7-yl)-ethanoneas an off-white solid.

¹H-NMR (CDCl₃) δ: 0.6-0.9 (6H, d), 1.2-1.6 (4H, m), 2.2-2.4 (4H, m),2.55 (3H, s), 2.8 (2H, d), 5.3 (2H, brs), 6.25 (1H, t), 6.85 (1H, d),7.27-7.4 (6H, m), 7.6-7.8 (2H, m), 8.0 (1H, d), 8.5 (1H, d).

MS m/z: 517 (M+1).

Step 9

The product of step 8 (500 mg, 0.969 mmol), NaOH (2M in water, 4.84mmol, 2.42 mL), sodium hypochlorite (4% available chlorine, 3.6 mmol)and DME (10 vols, 5 mL) were charged to a 25 mL round bottom flask andstirred at room temperature overnight. After 12 hours, sodium bisulfite(5 mL, saturated aq solution) was added and the reaction extracted withethyl acetate (4×5 mL); the organic layers were combined and dried oversodium sulfate, filtered and evaporated under reduced pressure to yield500 mg (96% yield) of a yellow solid. The solid was dissolved in water(20 vols, 10 mL) and acidified with acetic acid to pH 6.15. Uponacidification, a cream-colored solid was precipitated; the solid wasfiltered and placed in a vacuum oven for about two days to afford thetitled compound.

¹H-NMR (CD₃OD) δ: 0.75 (s, 3H), 0.86 (s, 3H), 1.63 (d, 1H), 2.49-2.66(m, 2H), 2.70-2.89 (m, 2H), 2.99-3.23 (m, 5H), 5.10-5.50 (brs, 2H), 6.15(t, 1H), 6.75 (d, 2H), 7.25-7.31 (m, 2H), 7.39-7.47 (m, 2H), 7.71-7.81(m, 2H), 7.98 (d, 1H), 8.45 (d, 1H).

MS m/z: 519 (M+1).

Example 2

(R)-5-{3-[4-(4-Chloro-phenyl)-4-hydroxy-3,3-dimethyl-piperidin-1-yl]-propylidene}-5,11-dihydro-10-oxa-1-aza-dibenzo[a,d]cycloheptene-7-carboxylic acid Part1: (R)-4-(4-chloro-phenyl)-3,3-dimethyl-piperidin-4-ol

Racemic 4-(4-chloro-phenyl)-3,3-dimethyl-piperidin-4-ol (0.500 g, 2.086mmol) was dissolved in minimal hot isopropyl alcohol (ca. 5 mL). The hotsolution was filtered through a plug of cotton and transferred to asolution of (1S)-(+)-10-camphorsulfonic acid (0.484 g, 2.086 mmol) inisopropyl alcohol (ca. 3 mL). The mixture was stirred vigorously forseveral minutes, during which a thick precipitate formed, and allowed tocool to room temperature over 0.25 hour. The solids were removed bysuction filtration and dried in vacuo. The dried salt was dissolved inhot isopropyl alcohol (ca. 50 mL), filtered through a cotton plug, andallowed to slowly cool to room temperature, undisturbed, overnight. Thesolids that formed on cooling (95 mg, 19% of theoretical) were removedby suction filtration and shown by analytical HPLC to beenantiomerically pure. The salt was suspended in ethyl acetate andneutralized with sodium hydroxide (1 N). The homogenous organic phasewas washed with water and brine, dried over sodium sulfate, filtered anddried to afford R-4-(4-chloro-phenyl)-3,3-dimethyl-piperidin-4-ol.

¹H-NMR (CD₃OD, 300 MHz) δ: 0.73 (s, 3H), 0.85 (s, 3H), 1.42 (ddd, 1H),2.36 (d, 1H), 2.61 (ddd, 1H), 2.91 (br dd, 1H), 3.08-3.19 (m, 2H),7.26-7.32 (m, 2H), 7.44-7.50 (m, 2H).

MS m/z: 240 (M+1).

Part 2

The compound was prepared essentially as described in Steps 5-9 ofExample 1, but replacing(5)-4-(4-chloro-phenyl)-3,3-dimethyl-piperidin-4-ol with(R)-4-(4-chloro-phenyl)-3,3-dimethyl-piperidin-4-ol.

Example 3

racemic-5-{3-[4-(4-Chloro-phenyl)-4-hydroxy-3,3-dimethyl-piperidin-1-yl]-propylidene}-5,11-dihydro-10-oxa-1-aza-dibenzo[a,d]cycloheptene-7-carboxylic acid

The racemic material was prepared essentially as described in Steps 5-9of Example 1, but replacing(5)-4-(4-chloro-phenyl)-3,3-dimethyl-piperidin-4-ol with racemic4-(4-chloro-phenyl)-3,3-dimethyl-piperidin-4-ol.

Example 4 THP-1 Cell Membrane Preparation and Binding Assay

Membranes were prepared from THP-1 cells (ATCC. #TIB202). Cells wereharvested by centrifugation, washed twice with PBS (phosphate-bufferedsaline), and the cell pellets were frozen at −70 to −85° C. The frozenpellet was thawed in ice-cold lysis buffer consisting of 5 mM HEPES(N-2-hydroxyethylpiperazine-N′-2-ethane-sulfonic acid) pH 7.5, 2 mM EDTA(ethylenediaminetetraacetic acid), 5 μg/ml each aprotinin, leupeptin,and chymostatin (protease inhibitors), and 100 μg/ml PMSF (phenylmethane sulfonyl fluoride—also a protease inhibitor), at a concentrationof 1 to 5×10⁷ cells/ml. This procedure results in cell lysis. Thesuspension was mixed well to resuspend all of the frozen cell pellet.Nuclei and cell debris were removed by centrifugation of 400×g for 10minutes at 4° C. The supernatant was transferred to a fresh tube and themembrane fragments were collected by centrifugation at 25,000×g for 30minutes at 4° C. The supernatant was aspirated and the pellet wasresuspended in freezing buffer consisting of 10 mM HEPES pH 7.5, 300 mMsucrose, 1 μg/ml each aprotinin, leupeptin, and chymostatin, and 10μg/ml PMSF (approximately 0.1 ml per each 10⁸ cells). All clumps wereresolved using a minihomogenizer, and the total protein concentrationwas determined using a protein assay kit (Bio-Rad, Hercules, Calif., cat#500-0002). The membrane solution was then aliquoted and frozen at −70to −85° C. until needed.

Binding Assays utilized the membranes described above. Membrane protein(2 to 20 μg total membrane protein) was incubated with 0.1 to 0.2 nM125I-labeled MIP-1α with or without unlabeled competitor (MIP-1ÿ) orvarious concentrations of compounds. The binding reactions wereperformed in 60 to 100 μl of a binding buffer consisting of 10 mM HEPESpH 7.2, 1 mM CaCl2, 5 mM MgCl2, and 0.5% BSA (bovine serum albumin), for60 min at room temperature. The binding reactions were terminated byharvesting the membranes by rapid filtration through glass fiber filters(GF/B or GF/C, Packard) which were presoaked in 0.3% polyethyleneimine.The filters were rinsed with approximately 600 μl of binding buffercontaining 0.5 M NaCl, dried, and the amount of bound radioactivity wasdetermined by scintillation counting. The activities of test compoundsare reported in the Table.

Example 5 In Vivo Efficacy Model

An animal model of neutrophil recruitment in response to MIP-1α was usedto evaluate the biological/pharmacodynamic activity of the compounds.Compounds were administered to female Hartley guinea pigs orally (dosesranged from about 0.5 mg/kg to about 5.0 mg/kg) 30 minutes prior tointradermal injections of murine MIP-1α (100 pmol/site) or phosphatebuffered saline (PBS). Skin punch biopsies were taken 5 hours later andprocessed for myeloperoxidase (MPO) measurements. MPO activity was usedas an indicator for neutrophil recruitment to the injection site. Theresults are presented in the Table.

Example 6

(S)-5-{3-[4-(4-Chloro-phenyl)-4-hydroxy-3,3-dimethyl-piperidin-1-yl]-propylidene}-1-oxy-5,11-dihydro-10-oxa-1-aza-dibenzo[a,d]cycloheptene-7-carboxylicacid Example 7

(R)-5-{3-[4-(4-Chloro-phenyl)-4-hydroxy-3,3-dimethyl-piperidin-1-yl]-propylidene}-1-oxy-5,11-dihydro-10-oxa-1-aza-dibenzo[a,d]cycloheptene-7-carboxylicacid Example 8

racemic-5-{3-[4-(4-Chloro-phenyl)-4-hydroxy-3,3-dimethyl-piperidin-1-yl]-propylidene}-1-oxy-5,11-dihydro-10-oxa-1-aza-dibenzo[a,d]cycloheptene-7-carboxylicacid Reference Example

The Reference Example was prepared as described in WO 01/09138.

TABLE Inhibition of ¹²⁵I-MIP- 1α Binding to THP-1 Cell MembranesEfficacy: Guinea Pig Neutrophil Example (Ki (nM)) Recruitment (ED50(mg/kg)) 1 2.3 0.12 2 >1000 not determined 3 3 99% inhibition at 2.5mg/kg Reference 7.8 3.6 Example

The data presented in the Table demonstrate that Examples 1 and 3 havegreater oral bioavailability and efficacy in comparison to thestructurally related compound of the Reference Example. Examples 1 and 3also showed greater selectivity, compared to structurally relatedcompounds, when assayed on other G protein-coupled receptors and ionchannels.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A compound having the formula:

or a physiologically acceptable salt thereof, wherein R¹ is a halogen.2. The compound of claim 1 wherein R¹ is selected from the groupconsisting of chloro, fluoro and bromo.
 3. The compound of claim 2wherein R¹ is chloro.
 4. A pharmaceutical composition comprising thecompound of claim 1 and a physiologically acceptable carrier orexcipient.
 5. A compound having the structure:

or a physiologically acceptable salt thereof, wherein R¹ is a halogen.6. The compound of claim 5 wherein R¹ is selected from the groupconsisting of chloro, fluoro and bromo.
 7. The compound of claim 6wherein R¹ is chloro.
 8. A pharmaceutical composition comprising thecompound of claim 5 and a physiologically acceptable carrier orexcipient.
 9. A method of treating a disease selected from the groupconsisting of inflammatory arthritis, inflammatory demyelinatingdisease, atherosclerosis, arteriosclerosis, restenosis,ischemia/reperfusion injury, diabetes mellitus, psoriasis, inflammatorybowel diseases, rejection of a transplanted graft, graft versus hostdisease, allergy and asthma, comprising administering to a subject inneed thereof an effective amount of a compound of claim
 5. 10. Acompound having the formula:

or a physiologically acceptable salt thereof, wherein R¹ is a halogen.11. A pharmaceutical composition comprising the compound of claim 10 anda physiologically acceptable carrier or excipient.
 12. The compound ofclaim 10 wherein R¹ is selected from the group consisting of chloro,fluoro and bromo.
 13. The compound of claim 12 wherein R¹ is chloro. 14.A method of treating a disease selected from the group consisting ofinflammatory arthritis, inflammatory demyelinating disease,atherosclerosis, arteriosclerosis, restenosis, ischemia/reperfusioninjury, diabetes mellitus, psoriasis, inflammatory bowel diseases,rejection of a transplanted graft, graft versus host disease, allergyand asthma, comprising administering to a subject in need thereof aneffective amount of a compound of claim
 10. 15. A method of treating adisease selected from the group consisting of inflammatory arthritis,inflammatory demyelinating disease, atherosclerosis, arteriosclerosis,restenosis, ischemia/reperfusion injury, diabetes mellitus, psoriasis,inflammatory bowel diseases, rejection of a transplanted graft, graftversus host disease, allergy and asthma, comprising administering to asubject in need thereof an effective amount of a compound of claim 1.16. A method for treating an inflammatory disease, comprisingadministering to a subject in need thereof an effective amount of acompound of claim
 1. 17. The method of claim 16, wherein saidinflammatory disease is an acute inflammatory disease.
 18. The method ofclaim 16, wherein said inflammatory disease is a chronic inflammatorydisease.
 19. A method for treating an inflammatory disease, comprisingadministering to a subject in need thereof an effective amount of acompound of claim
 5. 20. The method of claim 19, wherein saidinflammatory disease is an acute inflammatory disease.
 21. The method ofclaim 19, wherein said inflammatory disease is a chronic inflammatorydisease.
 22. A method for treating an inflammatory disease, comprisingadministering to a subject in need thereof an effective amount of acompound of claim
 10. 23. The method of claim 22 wherein saidinflammatory disease is an acute inflammatory disease.
 24. The method ofclaim 22 wherein said inflammatory disease is a chronic inflammatorydisease.
 25. A method for treating inflammatory arthritis, comprisingadministering to a subject in need thereof an effective amount of acompound of claim
 1. 26. The method of claim 25 wherein saidinflammatory arthritis is selected from the group consisting ofrheumatoid arthritis, juvenile rheumatoid arthritis, ankylosingspondylitis, reactive arthritis, Reiter's syndrome, psoriatic arthritis,psoriatic spondylitis, enteropathic arthritis, enteropathic spondylitis,juvenile-onset spondyloarthropathy and undifferentiatedspondyloarthropathy.
 27. A method for treating inflammatory arthritis,comprising administering to a subject in need thereof an effectiveamount of a compound of claim
 5. 28. The method of claim 27 wherein saidinflammatory arthritis is selected from the group consisting ofrheumatoid arthritis, juvenile rheumatoid arthritis, ankylosingspondylitis, reactive arthritis, Reiter's syndrome, psoriatic arthritis,psoriatic spondylitis, enteropathic arthritis, enteropathic spondylitis,juvenile-onset spondyloarthropathy and undifferentiatedspondyloarthropathy.
 29. A method for treating inflammatory arthritis,comprising administering to a subject in need thereof an effectiveamount of a compound of claim
 10. 30. The method of claim 29 whereinsaid inflammatory arthritis is selected from the group consisting ofrheumatoid arthritis, juvenile rheumatoid arthritis, ankylosingspondylitis, reactive arthritis, Reiter's syndrome, psoriatic arthritis,psoriatic spondylitis, enteropathic arthritis, enteropathic spondylitis,juvenile-onset spondyloarthropathy and undifferentiatedspondyloarthropathy.
 31. A method for treating inflammatorydemyelinating disease, comprising administering to a subject in needthereof an effective amount of a compound of claim
 1. 32. The method ofclaim 31 wherein said inflammatory demyelinating disease is acuteinflammatory demyelinating disease.
 33. The method of claim 32 whereinsaid acute inflammatory demyelinating disease is selected from the groupconsisting of acute disseminated encephalomyelitis, Guillain-Barresyndrome and acute hemorrhagic leukoencephalitis.
 34. The method ofclaim 31 wherein said inflammatory demyelinating disease is chronicinflammatory demyelinating disease.
 35. The method of claim 34 whereinsaid chronic inflammatory demyelinating disease is multiple sclerosis orchronic inflammatory demyelinating polyradiculoneuropathy.
 36. A methodfor treating inflammatory demyelinating disease, comprisingadministering to a subject in need thereof an effective amount of acompound of claim
 5. 37. The method of claim 36 wherein saidinflammatory demyelinating disease is acute inflammatory demyelinatingdisease.
 38. The method of claim 37 wherein said acute inflammatorydemyelinating disease is selected from the group consisting of acutedisseminated encephalomyelitis, Guillain-Barre syndrome and acutehemorrhagic leukoencephalitis.
 39. The method of claim 36 wherein saidinflammatory demyelinating disease is chronic inflammatory demyelinatingdisease.
 40. The method of claim 39 wherein said chronic inflammatorydemyelinating disease is multiple sclerosis or chronic inflammatorydemyelinating polyradiculoneuropathy.
 41. A method for treatinginflammatory demyelinating disease, comprising administering to asubject in need thereof an effective amount of a compound of claim 10.42. The method of claim 41 wherein said inflammatory demyelinatingdisease is acute inflammatory demyelinating disease.
 43. The method ofclaim 42 wherein said acute inflammatory demyelinating disease isselected from the group consisting of acute disseminatedencephalomyelitis, Guillain-Barre syndrome and acute hemorrhagicleukoencephalitis.
 44. The method of claim 41 wherein said inflammatorydemyelinating disease is chronic inflammatory demyelinating disease. 45.The method of claim 44 wherein said chronic inflammatory demyelinatingdisease is multiple sclerosis or chronic inflammatory demyelinatingpolyradiculoneuropathy.
 46. A method for treating rheumatoid arthritis,comprising administering to a subject in need thereof an effectiveamount of a compound of claim
 1. 47. A method for treating rheumatoidarthritis, comprising administering to a subject in need thereof aneffective amount of a compound of claim
 5. 48. A method for treatingrheumatoid arthritis, comprising administering to a subject in needthereof an effective amount of a compound of claim
 10. 49. A method fortreating multiple sclerosis, comprising administering to a subject inneed thereof an effective amount of a compound of claim
 1. 50. A methodfor treating multiple sclerosis, comprising administering to a subjectin need thereof an effective amount of a compound of claim
 5. 51. Amethod for treating multiple sclerosis, comprising administering to asubject in need thereof an effective amount of a compound of claim 10.